We're getting into some sucrose gradient centrifugation applications in our lab in order to separate chloroplasts out from lysed cells. We were wondering - is there a simple way to detect (or even quantify) mitochondria to see if they have co-separated with the chloroplasts? A dye or something?
Thanks.
Based on my experience in working with intact cells, I think you can stain samples with DAPI and check for the presence of mitochondria under fluorescence microscopy. The mitochondria will appear as speckles even under a 100X objective but you can see them and count them. Of course, you will have control samples stained that can be either positive (if you know where in your centrifugation tube to look for them) or negative.
I would suggest to run a citrate synthase assay. It is cheap, quantitative and convenient.
Regards, Christian
You could use the suggested citrate synthase and another one for cytochrome c oxidase. With that you could asses the purity of your fraction and have an idea about the integrity of the external and internal membrane of the mitochondria. Use the release of CS with detergentes to determinate if the internal membrane is broken or not (if I do not remember bad Clark has one method published for that). And use the cytochrome c oxydase for the external membrane integrity. You can check my publication for bibliography and the methods Iglesias-Gonzalez et al. J. Neurosci. Meth. 2013 (https://www.researchgate.net/publication/233974800_A_simple_method_for_isolating_rat_brain_mitochondria_with_high_metabolic_activity_Effects_of_EDTA_and_EGTA)
Article A simple method for isolating rat brain mitochondria with hi...
There are mitochondria specific dyes like MitoTrackers (green and red) or JC-1 which are suitable for specific mitochondria staining. If you don't care about mitochondrial membrane potencial you can use MT Green, but in case of detecting active versus inactive organelles I'd suggest using JC-1 or MT Red.
- Badges
- Science topic
- Similar topics
- Geoscience
- Atmospheric Sciences
- Climate Science
- Climate Change